The present invention relates to uricase having an optimum pH in the acidic region (hereinafter referred to as acidic uricase), a process for production thereof, a method for the quantitative determination of uric acid in a sample by enzymatic reaction and a test composition suitable therefor.
Uricase is a known enzyme which catalyzes the hydrolysis of uric acid to allantoin, hydrogen peroxide and carbon dioxide. The enzyme is, therefore, useful for the determination of uric acid in blood, urine, and the like.
Known uricases have an optimum pH in the neutral or alkaline region. For example, the optimum pH of uricase produced by culturing an Alternaria tenuis strain is 7.0 [Arch. Microbiol., vol. 17. 255 (1952)]. Similarly, it is known that uricase having an optimum pH in the range of 8.5-9.5 may be produced from many microorganisms and the like. For example, uricase is recoverable from rat liver [Biochemistry, vol. 13, 888 (1974)], Aspergillus flavus [C.R. Acad. Sci., vol. 264, 2244 (1967)], Arthrobacter pascens [Biochem. Biophys. Acta, vol. 151, 54 (1968)], Alcaligenes eutrophus [Arch. Microbiol. vol. 60, 160 (1968)], Bacillus fastidiosus [Anal. Biochem., vol. 38, 65 (1970)], Nocardia alba [Japanese Patent Publication No. 7749/'76], Streptomyces sp. [Agric. Biol. Chem., vol. 33, 1282 (1969)], and Enterobacter cloacae [Japanese Published Unexamined Patent Application No. 11296/1979]. The optimum pH of uricase produced by culturing Trichosporon cutaneum is around pH 8.0.
It is known that uricase may be used for the determination of uric acid in a sample. According to the known method, uric acid is oxidized with uricase in the presence of oxygen to form allantoin, hydrogen peroxide and carbon dioxide. The amount of uric acid is then calculated by determining at least one of the products formed or, alternatively, by the amount of oxygen consumed in the enzymatic reaction.
In the prior methods, generally, the determination of uric acid is conducted by the determination of hydrogen peroxide formed by a colorimetric method. In that method, the hydrogen peroxide is reacted with a coloring reagent to form a pigment and the absorbancy of the reaction solution is measured.
In the most simple procedure, the enzymatic reaction and color developing reaction is carried out in one step.
The known method, however, suffers from the disadvantage that when the enzymatic reaction is carried out at neutral or alkaline pH, the color-forming reaction is subject to interference from bilirubin, etc. in the blood. While it is desirable to carry out the enzymatic reaction at an acidic pH since a coloring reagent which forms a pigment having high sensitivity in color development can be used, the known uricases do not have an optimum pH within the acidic region.